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. 2012 Jul;92(4):328–332. doi: 10.1016/j.tube.2012.03.001

Figure 3.

Figure 3

Apo-WhiB1 binds at a site overlapping the −35 element of the groEL2 promoter. (A) Radiolabeled fragments of Rv0439c-Rv0440 (groEL2) intergenic DNA (F1–4) were incubated with nitric oxide-treated WhiB1 before separation of protein-DNA complexes by electrophoresis. The DNA fragments are shown on the right. The transcript start (arrow) and region protected by apo-WhiB1 in DNase I footprints (hatched box) are indicated. Lanes 1 and 2, Rv0439c-Rv0440 (groEL2) intergenic region extending from −116 to +181 bp (F1); lanes 3 and 4, Rv0439c-Rv0440 (groEL2) sub-fragment extending from −116 to –16 bp (F2); lanes 5 and 6, Rv0439c-Rv0440 (groEL2) sub-fragment extending from −116 to +34 bp (F3); lanes 7 and 8, Rv0439c-Rv0440 (groEL2) sub-fragment extending from −15 to +181 bp (F4). Numbering is relative to the groEL2 transcript start. Lanes 1, 3, 5 and 7, no protein; lanes 2, 4, 6 and 8, nitric oxide-treated WhiB1 (20 μM). The locations of the free DNA species (F1–F4) and the WhiB1 complexes (WhiB1-groEL2) are indicated. (B) DNase I footprint of the groEL2 promoter: lane 1, no apo-WhiB1, lanes 2 and 3, apo-WhiB1 (20 μM), lane M, Maxam and Gilbert G track. The region of DNA protected from DNase I digestion is indicated by the open rectangle; a hypersensitive site (−27) is arrowed. The closed rectangles indicate the locations of the −35 and −10 elements and the transcript start is marked by +1. The DNA sequence of the groEL2 promoter is shown on the right. The region of apo-WhiB1 protection is boxed, the −35 and −10 elements are underlined and the transcript start (+1) is in bold type.